Cells in multicellular organisms sense and respond to their environments is integral to the formation of tissues and organs in development, adaptation to changes in nutrient levels and hormone signals, and regeneration of damaged tissues in adults. Lesions in these sensing systems result in disease states such as birth defects, cancer, and degenerative diseases. Thus, understanding the molecular mechanisms behind cell sensing will not only provide a better understanding of biological processes, it will provide insights into the causes of human diseases, and will aid diagnosis, treatment, and cure. My laboratory has been studying one such pathway, the Notch signaling pathway. In Notch signaling, local information from neighboring cells is sensed through a transmembrane receptor encoded by the Notch gene(s), and is transduced into the nucleus to activate the expression of downstream genes, leading to cell differentiation. One unique feature of the Notch pathway is that upon activation, the receptor is converted to a transcription factor through cleavage of the Notch intracellular domain (NICD) from the membrane. Following cleavage, NICD activates transcription by binding a factor called CSL. This interaction involves bivalent contacts between distant regions of NICD and CSL, and is mediated by a ~100 residue RAM linker segment, which though largely disordered, plays a key role in orchestrating displacement of co-repressors, and recruitment of coactivators. NICD activity is also strongly modulated by a number of cytosolic proteins such as Deltex, although the mechanistic details are murky. In this proposal, we use traditional biophysical, structural methods, and solution thermodynamics to characterize these aspects of Notch signaling, but also combine these methods with functional assays for Notch signaling using mammalian cell culture. This second approach is a new direction for my laboratory, but results so far have been rewarding, giving results from biophysical studies a functional context, and focusing cell culture studies on quantitative, sharply focused mechanistic questions. We are also putting greater emphasis on NMR and computational simulations. Specifically, we are determining the structural basis of interaction between the Notch receptor and Deltex, are identifying surface substitutions that disrupt binding, and are determining how disrupting this interaction perturbs ubiqutination, transcriptional activation, receptor endocytosis, and interaction with a putative deubiquitinase. In addition, we are characterizing the RAM region of NICD using NMR, AUC, simulation, and mutagenesis. Finally, we are determining how RAM enhances the conserved bivalent interaction between NICD and CSL, and how this bivalent interaction thermodynamically couples corepressor dissociation with coactivator binding. The system under study here is ideal for understanding how intrinsically disordered regions influence protein structure and function.